Project Summary The skin not only serves as a physical barrier, but also harbors a variety of innate and adaptive immune cell types that collectively maintain a functional barrier against the invasion of commensal microbes and environmental pathogens. This barrier function has to be balanced with the need to avoid exaggerated or inappropriate immune responses that can induced skin immunopathology. CD8+ epidermal resident memory T cells (eTrm) have recently become recognized as a critical component of skin immune defense. However, in addition to protecting us against re-infection with previously encountered pathogens, they likely also play important pathogenic roles in several autoimmune and inflammatory skin diseases, such as psoriasis, contact dermatitis, chronic eczema, and others. Understanding how eTrm are formed and maintained is therefore relevant to the development of novel therapeutic strategies for skin disease. Genetic mouse models have been highly instructive in identifying key proteins and molecular mechanisms that mediate eTrm differentiation in various epithelial tissues, including skin. However, they have not yet produced a comprehensive model describing the sequential migratory steps, by which during an inflammatory response some effector T cells in are retained in skin, guided to cross the epithelial basement membrane to enter the epidermis, and settle into niches that promote the capacity for long-term local persistence as local memory cells. This has hindered an integrated understanding of this process, which will be required for the rational design of therapeutic interventions to either enhance eTrm formation in the context of vaccination or disrupt their formation in the context of skin immunopathology. He we propose to develop a multiphoton intravital microscopy approach to visualize and comprehensively characterize migratory events than facilitate the process of eTrm differentiation in mouse skin. We will assess how CD8 T cells that are insensitive to the cytokine TGF-beta diverge in their migratory behavior from wildtype cells, and use fluorescent fusion protein reporter constructs to visualize in real time how T cell instantaneously collect TGF-beta signals and thereby explore the context in which these critical signaling events occur. Finally, we will determine the function of the TGF-beta-regulated genes CD103 and S1P-receptor-1 by testing their ability to restore aspects of eTrm differentiation in TGF-beta-insensitive T cells.